This web page was produced as an assignment for an undergraduate course at Davidson College.

The Effect of Microbiota on Growth of Juvenile Mice

L. plantarum. Image Courtesy of Kenyon College

This paper focused on the microbiota’s role in maintaining appropriate growth rates in juvenile mice.The authors, in particular, discovered that specific strains of Lactobacillus plantarum have the ability to either stunt (Lp^NIZO2877) or maintain growth (Lp^WJL) even if the organism being studied is experiencing malnutrition. This was done through the direct comparison of wild type and germ free mice for 8 weeks after birth, comparing various hormone levels relating to the somatotropic axis and bone characteristics. The researchers reported significantly decreased growth hormone and growth factor levels in the germ free mice each time the levels were tested throughout the 56 days that they were studied. These tests were first done on mice that were fed on a standard breeding diet. Following those tests, the researchers then turned to a chronic undernutrition standard and followed mice who had been weaned to a nutritionally depleted diet. The results for this diet were that, after an initial weight loss period for both wild type and germ free groups, the wild type resumed their growth, even though the level was depleted in comparison to the standard breeding diet. The germ free mice had a stunted growth that was not due to eating less than the wild type mice on the same diet. The researchers saw similar decreases in the various hormone levels that they tested in the germ free mice when compared to the wild type mice for this depleted diet. The researchers then used Drosophila to identify the two strains of L. plantarum, and colonized mice with either Lp^WJL or Lp^NIZO2877. They saw that the Lp^WJL strain had the ability to promote a wild type-like growth in mice, while the Lp^NIZO2877 strain was more comparable to the germ free mice. Finally the researchers treated mice with either a growth factor inhibits or DMSO for a 10 day period to confirm that microbiota combats the effect that undernutrition has on the activity of the somatotropic axis. The authors close the paper with a suggestion that this could be developed into a treatment for combating chronic undernutrition in children under the age of 5.

Figures:

Figure 1:

This figure is used to display the implications that the microbiota is relevant for maintaining juvenile growth. Panel A shows the weight trends of both wild type and germ free mice over the eight weeks that they were followed and shows a significant decreased weight for the germ free mice at every measurement. Furthermore, this panel showed a more pronounced difference after weaning. Panel B shows the averaged daily weight gain for both wild type and germ free mice, with a statistically significant higher weight per day for the wild type mice. Panel C shows the overall body length trends for both wild type and germ free mice, showing a significantly smaller body length for the germ free mice in comparison to the wild type. Panel D is similar to Panel B, but focuses instead on daily body length increases of both the wild type and germ free mice. This again shows a significant increase in the wild type growth rate in comparison to the germ free. Panel E shows pictures of a femur bone from a wild type mouse and a germ free mouse on day 56, with the wild type bone appearing to be the larger of the two. Panel F shows a cross section of distal parts of femur bones from both wild type and germ free individuals on day 56, and shows the germ free bone to appear thinner and less dense.

Figure 2:

This figure is used to confirm the maintenance of the activity of the somatotropic axis by the microbiota by comparing specific levels for various growth hormones and growth factors and their expression in wild type and germ free mice. Panel A focuses on the levels of Growth Hormone (GH) that are seen in both germ free and wild type mice throughout the study. The levels were seen to be highest around birth and gradually decreased throughout the study, but were not different in either type of mouse. Panel B focuses on the levels of IGF-1 (insulin-like growthfactor-1) in both kinds of mice throughout the study. Significantly lower levels were seen in germ free mice across the study. Panel C focuses on thelevels of IGFBP-3 (IGF-1 Binding Protein 3) in both mice halfway through (28 days) and at the end of the study (56 days). The relative levels were significantly higher for the wild type mice at both measurements. Panel D and E quantity the expression levels of Igf1 and Igfbp3 in the livers of both germ free and wild type mice. The expression levels of both genes were significantly decreased in germ free mice. Panel F focuses on the phosphorylation of Akt at Ser 473, which can be used as an indication of IGF-1receptor signaling activity. This shows western blots for both wild type and germ free mice which it then quantifies. Significantly more phosphorylation is seen in wild type mice.

Figure 3:

This figure focuses on the strain specificity of L. plantarum and its impacts on juvenile growth in mice. Panel A shows the weight trends of mice that were either wild type, germ free, or colonized with either Lp^WJL or Lp^NIZO2877 and were fed either a standard breeding diet or a depleted diet. This shows that mice colonized with Lp^WJL had higher body weights, comparable to wild type levels, than the Lp^NIZO2877  mice did. Furthermore, this difference between the strains maintained even with a depleted diet, suggesting the role of the microbiota can combat the effects of undernutrition. Panel B shows photographs of a mouse from each type on the final day of study. The Lp^WJL mice appear to be comparable in size to the wild type mice shown while the Lp^NIZO2877 appear to be similar in size to the germ free mice. Panel C does the same comparisons that are seen in Panel A but with body length instead of weight. This shows that mice colonized with Lp^WJL had longer bodies, comparable to wild type lengths, than the Lp^NIZO2877  mice did, regardless of what diet they were fed on. Panel D shows photographs of the femurs of all four types of mice. This shows that there is a difference in bone size between the two strains of mice, with the Lp^NIZO2877  mice having notably smaller femurs than Lp^WJL mice had, which appear identical to the wild type femurs.

Figure 4:

This figure focuses on the somatotropic axis activity levels in mice undergoing undernutrition.Panels A focuses on Growth Hormone levels in mice that were either wild type, germ free, or colonized with either Lp^WJL or Lp^NIZO2877 and were fed a depleted diet. A significant increase can be seen in the Lp^NIZO2877  levels for when compared to either the wild type or the Lp^WJL strain at the halfway measurement. Furthermore, Lp^WJL mice show a significant decrease when compared to the germ free mice at the 28 day measurement. No significance can be seen between all four levels at the end of the study, which is to be expected as Growth Hormone levels decrease over time. Panel B focuses on IGF-1 levels in mice that were either wild type, germ free, or colonized with either Lp^WJL or Lp^NIZO2877 and were fed a depleted diet. Lp^NIZO2877 shows a significantly decreased level when compared to either Lp^WJL  or wild type mice at both measurements (28 days and 56 days). Furthermore, a significant decrease can be seen in the IGF-1 levels for germ free mice when compared to either Lp^WJL  or wild type mice at the 56 day study. Panel C focuses on IGFBP-3 levels in mice that were either wild type, germ free, or colonized with either Lp^WJL or Lp^NIZO2877 and were fed a depleted diet. A significant increase in IGFBP-3 levels can be seen for Lp^WJL mice when compared to either wild type or germ free mice at the 28 day measurement. By 56 days, a significantly increased level could be seen in Lp^WJL mice compared to Lp^NIZO2877 mice and germ free mice. Furthermore, a significant decrease could be seen between Lp^WJL, Lp^NIZO2877, and germ free mice when compared to wild type mice. Panels D-G focus on wild type mice that were on either a standard breeding diet or a depleted diet and were treated with either DMSO or PPP, which is an IGF-1R inhibitor. Panel D showsthat a significant difference in daily weight gain can be seen in mice fed a breeding diet who were treated with PPP when compared to those on the same dieswho were treated with PPP. No difference can be seen between either treatment in the depleted diet. Panel E shows that a significantly increased body length gain can be seen in mice treated with DMSO on a breeding diet compared to those on the same diet treated with PPP. No difference can be seen between either treatment in the depleted diet. Panel F focuses on differences in femur length seen for both treatments in breeding diet mice. The femurs of DMSO treated mice were significantly longer than those treated with PPP. Panel G focuses on differences in femur length seen for both treatments in depleted diet mice. The femurs of PPP treated mice were significantly shorter than those treated with DMSO.

I felt that this paper did a good job at making its arguments convincing while still being concise. I also felt like the authors supported their future applications of this microbiota study by utilizing strains that had been identified in a different model organism and effectively showing their role in mice. Furthermore, their figures were, for the most part, easy to read. The main problem that I had was having a hard time reading the markers for distinguishing statistical significance. I think that a future progression of this in a variety of organisms would allow for a better determination of the implications for medicine.

References

1. Schwarzer M. et al. 2016. Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science. 351 (6275) 854-857.